Hybridized quantum dot, silica, and gold nanoparticles for targeted chemo-radiotherapy in colorectal cancer theranostics

Multimodal nanoparticles, utilizing quantum dots (QDs), mesoporous silica nanoparticles (MSNs), and gold nanoparticles (Au NPs), offer substantial potential as a smart and targeted drug delivery system for simultaneous cancer therapy and imaging. This method entails coating magnetic GZCIS/ZnS QDs with mesoporous silica, loading epirubicin into the pores, capping with Au NPs, PEGylation, and conjugating with epithelial cell adhesion molecule (EpCAM) aptamers to actively target colorectal cancer (CRC) cells. This study showcases the hybrid QD@MSN-EPI-Au-PEG-Apt nanocarriers (size ~65 nm) with comprehensive characterizations post-synthesis. In vitro studies demonstrate the selective cytotoxicity of these targeted nanocarriers towards HT-29 cells compared to CHO cells, leading to a significant reduction in HT-29 cell survival when combined with irradiation. Targeted delivery of nanocarriers in vivo is validated by enhanced anti-tumor effects with reduced side effects following chemo-radiotherapy, along with imaging in a CRC mouse model. This approach holds promise for improved CRC theranostics.

1. Multimode imaging sounds fancy, but what would be the rationale to include multiple imaging if one imaging modality works? 2. When investigating the cell death mechanism in vitro, did you confirm that tumor cells did not die through mechanisms other than apoptosis, like necrosis, autophagy, or immunogenic cell death?3. The in vivo investigation of tumor cell apoptosis needs to be provided.4. Tumor targeting in vivo should be tested.5.There are many inorganic NPs on this platform.What would be the elimination pathways?6. Advantages and disadvantages of this approach should be discussed, as well as future investigations.7. The scale bars in Figure 4 need to be marked more clearly 8.The overall figure presentation can be improved.
Reviewer #3 (Remarks to the Author): In this study, the authors employ multimodal theranostic nanoparticles (QD@MSNs-EPI-Au-PEG-Apt) for targeted drug delivery to CRC cells.This strategy commendably solves the deficiencies of poor targeting and side effects of traditional chemotherapy drugs.Moreover, this designed theranostic nanoparticles not only exhibited a significant therapeutic efficiency in the HT-29 tumor-bearing mice but also could be utilized for FL, MR, and CT imaging to visualize the biodistribution of QDs and Au NPs.Although the novelty of this work is limited, the experiments are well organized with lots of data.This manuscript could be accepted for publication provided that the authors addressed the following issues: Major comments 1.The stability of QD@MSNs-EPI-Au-PEG-Apt in vitro should be evaluated.2. On page 31, line 698, the authors indicate that no toxicity related to QD@MSN as backbone was observed on HT-29 and CHO cells， but there is no free QD@MSN group in Fig 5A and B. Please explain it.3. In the manuscript, the description of Figures 6A and B is missing.

Reviewer #1
The present manuscript has demonstrated the development of multimodal nanoparticles, incorporating quantum dots coated with mesoporous silica, capped with gold nanoparticles, aiming to offer targeted drug delivery in colorectal cancer.In vitro and in vivo studies have been conducted to examine aptamer-mediated CRC-specific cytotoxicity with radiosensitization, and imaging capability through MRI and CT methods, with the potential to provide a theranostic solution.Overall, the results are interesting.However, there are some specific concerns that needs to be addressed in major revision before recommending the manuscript for publication.My specific comments are appended below.
Comment 1: The authors are encouraged to shed light on the optical properties of free epirubicin by providing spectral data (absorption and fluorescence emission spectra).This will aid in understanding how epirubicin enhances the fluorescence when encapsulated into the quantum dots.
Response: Thank you for bringing this to our attention.We added the absorption and fluorescence emission spectra of free EPI and QD@MSN-EPI to the results as shown in Fig. 1E and F (shown below).The results confirmed the enhancement of both absorption and fluorescence emission by combination of QDs and EPI.
Comment 2: Multiple images in Figure 1A panel should be properly renumbered and mentioned in the legend and manuscript text to avoid confusion about multiple images under a single notation.
Response: With many thanks, Figure 1 was renumbered and different parts are mentioned in the legend and manuscript text (shown above).
Comment 3: The authors are encouraged to report the exact peak maxima of the Au NPs.

Response:
We are grateful for your careful comment, it was mentioned in the passage line 426 (517.26nm) in the section 3.1, as highlighted.
"As shown in Fig. 1H, the UV-Visible spectrum of as-prepared Au NPs revealed a maximum peak of 517.26 nm due to surface plasmon resonance (SPR) absorption" Comment 4: Reporting of hydrodynamic diameter and polydispersity index of quantum dots is essential.
Response: Respected reviewer, we would like to further elaborate on the issue regarding the assessment of the hydrodynamic diameter of QDs.Due to their hydrophobic nature, relying solely on the hydrodynamic diameter would not provide an accurate evaluation.In line with the reference article, we conducted size evaluations after the phase transfer of GZCIS/ZnS QDs to the aqueous phase using BSA.The resulting hydrodynamic diameter was found to be approximately 32.05 ± 1.43 (Guo et al. 2014).Additionally, we assessed the hydrodynamic diameter of the QDs after phase transition using CTAB, which yielded a size of approximately 8.43 ± 2.67.Despite having these data, we chose to report the QDs' size using HR-TEM, as it is considered more reliable and precise, yielding an approximate size of 4 nm.To better address the size of the QDs using dynamic light scattering (DLS), we presented the QDs stabilized by CTAB as the closest approximation to bare QDs, as shown in Table 1 below.

Reviewer #2
The authors reported a multimodal nanocarrier for targeted cancer treatment.While the concept might be interesting, there are several major issues that should be addressed.
Comment 1: Multimode imaging sounds fancy, but what would be the rationale to include multiple imaging if one imaging modality works?
Response: We are grateful for your careful comment.While in some points we introduced the importance of multiple imaging in the introduction (highlighted), we expanded the reasons of developing multimodal probes for different imaging modalities in discussion as highlighted in lines 994-999.
Comment 2: When investigating the cell death mechanism in vitro, did you confirm that tumor cells did not die through mechanisms other than apoptosis, like necrosis, autophagy, or immunogenic cell death?
Response: Respectfully, based on the reference articles, we anticipated that apoptosis and necrosis would be the primary cell death mechanisms.To confirm this, we conducted Annexin V-FITC/PI staining on the treated cells and analyzed them using flow cytometry.Our observations revealed an increase in apoptotic cell populations, suggesting enhanced early and late apoptosis, as well as minimal necrosis following the treatments.Conducting further specific experiments would be beneficial to determine the precise mechanism.However, it is important to note that extensive studies have already been conducted on the individual components of our therapeutic approach in relation to the aforementioned cell death mechanisms.Response: With utmost respect, we added fluorescence microscopy of sectioned tumor tissues for this matter in section 3.10 and Fig. 8G (shown below) which the results were in line with similar studies (Bredlau et al. 2018;Cui et al. 2017;M. W. Kim et al. 2017;H. Kim et al. 2019)."The merged fluorescence microscopy images of tumor sections, combining emissions from DAPI, EPI, and QDs, clearly showed a significant uptake of targeted nanoparticles in the tumor cells of the mice that were treated".
Overall, we aimed to elucidate the tumor targeting ability of nanoparticles using three imaging modalities: fluorescence (FL), magnetic resonance (MR), and computed tomography (CT) imaging.These imaging techniques were employed to examine and compare the localization and distribution of different nanoparticles in the mouse body, allowing us to evaluate the efficacy of targeted nanoparticles in selectively accumulating within the tumor compared to non-targeted nanoparticles.In addition, we extensively evaluated the anti-tumor efficacy of the targeted nanocarrier, further confirming the successful tumor targeting in vivo.
Comment 5: There are many inorganic NPs on this platform.What would be the elimination pathways?
Response: We greatly appreciate your consideration of this point.While it is important to emphasize the need for further investigations in the future, our current imaging results have provided significant insights.
We have observed a gradual decrease in intensity in both organs and tumors after the second time point, indicating a potential elimination of nanoparticles.Based on the importance of this issue we mentioned about the elimination process in line 982-986.It is worth noting that our study aimed to demonstrate the potential of hybridized nanoparticles for combinational treatment and imaging modalities, taking into account the previous investigations on the elimination pathways of each component (as described below and highlighted in line 990-998 of manuscript).
Studies have revealed that inorganic nanoparticles larger than 6 nm are typically cleared from the body through hepatobiliary and feces excretion, while smaller nanoparticles with sizes below 5.5 nm are rapidly metabolized and excreted through the urinary system (Yu and Zheng 2015).Specifically, numerous studies have focused on the renal clearance of QDs and have found that nanoparticles with a hydrodynamic size (HD) of ≤5.5 nm are efficiently excreted through urine, resulting in their effective clearance (Huang et al. 2007).
Amorphous silica has been found to be a biocompatible material for coating the surface of QDs in biomedical applications, improving their overall biocompatibility by preventing the release of QD components (Ma et al. 2010).Various research groups have investigated the biocompatibility and degradability of silica nanoparticles in vitro and in vivo, focusing on factors such as size, morphology, and surface functionalization (Kempen et al. 2015).Researchers, like Yamada et al., have studied colloidal mesoporous silica nanoparticles (CMPS) of different sizes and observed that regardless of size, more than 90% of the nanoparticles degraded after one week, with approximately 15% of the CMPS framework degrading daily (Yamada et al. 2012).Similarly, He et al. investigated the in vivo biodistribution and excretion of MSNs (mesoporous silica nanoparticles) with different sizes (80, 120, 200, and 360 nm) over a period of one month.They also examined the impact of PEGylation on MSNs.The results showed that PEGylation slightly reduced the uptake of MSNs in the liver, spleen, and lungs, leading to a longer bloodcirculation half-life and slower biodegradation and excretion compared to bare MSNs of the same sizes.
Smaller MSNs were found to evade capture by the liver and spleen more effectively, slowing down their degradation and excretion.The majority of MSNs were excreted through urine and feces, and the rate of clearance was influenced by the shape of the MSNs, with long-rod MSNs exhibiting a slower clearance rate than short-rod MSNs (He et al. 2011).
AuNPs, which are non-biodegradable nanoparticles, with a size smaller than 5-10 nm, can be cleared from the body through renal filtration.These particles are filtered by the kidneys and eliminated in the urine.According to proposed in vivo decision tree by Poon et al., nanoparticles below the glomerular filtration size limit (about 5.5 nm) are typically eliminated through the kidneys (Poon et al. 2019).They also observed fecal elimination for these small nanoparticles.On the other hand, larger non-biodegradable nanoparticles or biodegradable nanocarriers may undergo disassembly, breakdown, or metabolism and either return to the systemic circulation or get retained in Kupffer cells in the liver (Poller et al. 2018).In some cases, if Kupffer cells are avoided or incapacitated, nanoparticles may undergo hepatobiliary elimination (Sadauskas et al. 2007).
Comment 6: Advantages and disadvantages of this approach should be discussed, as well as future investigations.
Response: We sincerely appreciate your thoughtful comment.Based on your suggestion, we have made improvements to the structure of the paper.The advantages and disadvantages have been included directly in the last paragraph of the discussion.Additionally, the future investigations have been mentioned in the final sentence of the conclusion.
The main anticancer component loaded in nanocarrier (epirubicin) is well known to induce cell death in cancer cells primarily through the apoptotic pathway.Epirubicin belongs to a class of chemotherapy drugs called anthracyclines, which have a broad spectrum of anticancer activity.Epirubicin exerts its cytotoxic effects by intercalating with DNA and inhibiting topoisomerase II activity, resulting in DNA damage and impaired DNA repair mechanisms.This ultimately triggers signaling pathways that lead to apoptosis in cancer cells (Liu et al. 2017).On the other hand, radiotherapy could lead to apoptosis as described by references 101, 102 and 103 in the manuscript in lines 932-939 (6 MV X-ray in the presence of Au NPs).As demonstrated by Yang et al., the combinational effects of chemotherapy and radiation therapy of Au NPs led to DNA disfunction as the main inducible factor for apoptosis (Yang et al. 2018).More specifically, Terranova-barberio et al. evaluated the synergistic effects of chemotherapy and radiotherapy on apoptosis of HT-29 cells (Terranova-barberio et al. 2017).Comment 3: The in vivo investigation of tumor cell apoptosis needs to be provided.Response: With utmost gratitude for your suggestion, we conducted additional experiments to assess apoptosis at the tumor level.Specifically, we utilized the TUNEL staining technique to detect DNA fragmentation, a characteristic feature of apoptotic cells.The results obtained from these experiments have been incorporated into Fig.6F (shown below), and explained in section 3.8 of the manuscript.Comment 4: Tumor targeting in vivo should be tested.

Comment 7 :
The scale bars in Figure 4 need to be marked more clearly Response: Thank you for your valuable feedback on the scale bars in Fig. 4.Although there might be a slight decrease in quality when converting the image to Word and PDF formats, we want to assure you that we have made every effort to enhance the clearity of the image and scale bars.Comment 8: The overall figure presentation can be improved.Response: With respect, some figures such as Fig. 1, Fig. 4, Fig. 6 and Fig. 8 were improved.

Table 1
Mean values of size, PDI, and zeta potential of synthesized formulations in this